Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Science 29 August 2003:
Vol. 301. no. 5637, pp. 1235 - 1238
DOI: 10.1126/science.1084387
Prev | Table of Contents | Next

Reports

Single-Molecule Kinetics of {lambda} Exonuclease Reveal Base Dependence and Dynamic Disorder

Antoine M. van Oijen,1 Paul C. Blainey,1 Donald J. Crampton,2 Charles C. Richardson,2 Tom Ellenberger,2 X. Sunney Xie1*

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of {lambda}-phage DNA by individual bacteriophage {lambda} exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.

1 Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA. 2 Department of Biological Chemistry and Pharmacology, Harvard Medical School, 240 Long-wood Avenue, Boston, MA 02115, USA.

* To whom correspondence should be addressed. E-mail: xie{at}chemistry.harvard.edu

Read the Full Text


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression.
N. Y. Yao, R. E. Georgescu, J. Finkelstein, and M. E. O'Donnell (2009)
PNAS 106, 13236-13241
   Abstract »    Full Text »    PDF »
See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins.
J. van Mameren, E. J. G. Peterman, and G. J. L. Wuite (2008)
Nucleic Acids Res. 36, 4381-4389
   Abstract »    Full Text »    PDF »
Electrochemical detection of low-copy number salivary RNA based on specific signal amplification with a hairpin probe.
F. Wei, J. Wang, W. Liao, B. G. Zimmermann, D. T. Wong, and C.-M. Ho (2008)
Nucleic Acids Res. 36, e65
   Abstract »    Full Text »    PDF »
Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis.
M. Morimatsu, H. Takagi, K. G. Ota, R. Iwamoto, T. Yanagida, and Y. Sako (2007)
PNAS 104, 18013-18018
   Abstract »    Full Text »    PDF »
Single phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability.
D. N. Fuller, D. M. Raymer, V. I. Kottadiel, V. B. Rao, and D. E. Smith (2007)
PNAS 104, 16868-16873
   Abstract »    Full Text »    PDF »
From the Cover: NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork.
W. Cheng, S. Dumont, I. Tinoco Jr., and C. Bustamante (2007)
PNAS 104, 13954-13959
   Abstract »    Full Text »    PDF »
Single-molecule Chemistry and Biology Special Feature: Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase.
G. Luo, M. Wang, W. H. Konigsberg, and X. S. Xie (2007)
PNAS 104, 12610-12615
   Abstract »    Full Text »    PDF »
Processive enzyme mimic: Kinetics and thermodynamics of the threading and sliding process.
R. G. E. Coumans, J. A. A. W. Elemans, R. J. M. Nolte, and A. E. Rowan (2006)
PNAS 103, 19647-19651
   Abstract »    Full Text »    PDF »
Real-time Enzyme Dynamics Illustrated with Fluorescence Spectroscopy of p-Hydroxybenzoate Hydroxylase.
A. H. Westphal, A. Matorin, M. A. Hink, J. W. Borst, W. J. H. van Berkel, and A. J. W. G. Visser (2006)
J. Biol. Chem. 281, 11074-11081
   Abstract »    Full Text »    PDF »
A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA.
P. C. Blainey, A. M. van Oijen, A. Banerjee, G. L. Verdine, and X. S. Xie (2006)
PNAS 103, 5752-5757
   Abstract »    Full Text »    PDF »
Specificity of the Endonuclease Activity of the Baculovirus Alkaline Nuclease for Single-stranded DNA.
V. S. Mikhailov, K. Okano, and G. F. Rohrmann (2004)
J. Biol. Chem. 279, 14734-14745
   Abstract »    Full Text »    PDF »
Protein Conformational Dynamics Probed by Single-Molecule Electron Transfer.
H. Yang, G. Luo, P. Karnchanaphanurach, T.-M. Louie, I. Rech, S. Cova, L. Xun, and X. S. Xie (2003)
Science 302, 262-266
   Abstract »    Full Text »    PDF »


To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)